Abstract

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.

Different classes of proteins from Xenopus egg extract are retained by a CRM1 column. Eluates of a z-tagged CRM1 IgG-Sepharose column that had been incubated with Xenopus egg extract in the absence (lane 1) or presence (lanes 2 and 4) of 2 μM RanQ69LGTP and/or 50 μM MVM NS2 NES peptide (lanes 3 and 4) were separated on a gradient SDS–5 to 20% polyacrylamide gel and visualized by silver staining. Positions of bound X92, X94, X280, and RanQ69LGTP are indicated. A molecular mass (MM) marker (in kilodaltons) is shown at the right.

An3 is a shuttling DEAD-box helicase. (A) A conserved N-terminal NES sequence in the An3-Ded1p family of RNA helicases is revealed by alignment of the amino termini of Xenopus An3 (), human DBX and DBY (), mouse ERH () and PL10 (), and S. cerevisiae Ded1p () and Dbp1p (). The proposed leucine-rich NES consensus sequence is shown at the bottom (, ). The Φ sign denotes amino acids M, V, I, L, F, or W. (B) An3 binds to CRM1 via its extreme N terminus. A mixture of in vitro-translated 35S-labelled An3 and An3Δ21 (lane 1) were incubated with a z-tagged CRM1 column in the presence of 2 μM RanGTP and in the absence (−) or presence of 0.4 mM NS2 NES peptide (NS2) or a mutated (mut.) peptide as indicated. Bound (lanes 5 to 7) or flowthrough (lanes 2 to 4) fractions were analyzed by SDS-PAGE and fluorography. Positions of An3 and An3Δ21 are indicated at the right. (C) An3 is exported by the CRM1 pathway. A mixture of in vitro-translated 35S-labelled An3 (lanes 1 to 10) or An3Δ21 (lanes 11 to 14) and [γ-35S]ATP-labelled GST was microinjected into Xenopus oocyte nuclei in the presence of 0, 0.1, or 1 mM NS2 NES peptide or a mutated NES peptide (mut) as indicated. GST forms a multimer that remains in the compartment of injection to serve as injection and dissection control. Oocytes were dissected into nuclear (N) and cytoplasmic (C) fractions immediately (lanes 1 to 2 and 11 to 12) or after an incubation of 90 min (lanes 3 to 10 and 13 to 14), and labelled proteins were visualized by SDS-PAGE and fluorography. Positions of An3, An3Δ21, and GST are indicated. (D) An3 is imported into nuclei of Xenopus stage V and VI oocytes. The same mixture of proteins as described for panel C, as indicated, was microinjected into the cytoplasm of Xenopus oocytes that were dissected into nuclear (N) and cytoplasmic (C) fractions after 0 (lanes 1 to 2 and 5 to 6), 8 (lanes 7 to 8), or 22 h (lanes 3 to 4 and 9 to 10). (E) Nuclear accumulation of An3 in stage IV oocytes. Radiolabelled wild-type (wt) or Leu-19/21-Ala (mut) An3 were injected together with [35S]GST into the cytoplasm of stage IV oocytes and dissected as described above at t = 0 or after 8 h. Nuclear accumulation was quantified by with phosphorimager analysis after SDS-PAGE.

Quantitative analysis of NES-CRM1 affinity in vitro using the CRM1 GAP assay. (A) Comparison between CRM1-NES affinities as measured from CRM1-dependent protection of Rna1p-stimulated GTP hydrolysis on Ran, as a function of increasing concentrations of peptides representing wild-type or mutated MVM NS2 NES, HIV-1 Rev NES, protein kinase inhibitor (PKI) NES, or An3 NES. In all series, CRM1 is present at 100 nM, except series marked “(−),” where the effect of MVM NS2 wild type is measured in the absence of CRM1; the RanGTP concentration in all reaction mixtures is 200 pM. (B) Comparison between CRM1-dependent protection of RanGTP hydrolysis in the presence or absence of 1 μM HIV-1 Rev or GST-An3N as a function of increasing concentrations of CRM1.

An NES peptide that functions as a specific CRM1 inhibitor in vivo. A mixture of 32P-labelled in vitro-transcribed ftz pre-mRNA, U1ΔSm snRNA, U6Δss snRNA, and initiator methionyl-tRNA (tRNAmeti) were injected into Xenopus oocyte nuclei in the presence of 0, 0.1, or 1 mM MVM NS2 NES peptide (wt) or a mutated version thereof (mut) as indicated above the lanes. After 0 (lanes 1 and 2) or 90 min (lanes 3 to 10), nuclear (N) and cytoplasmic (C) fractions were obtained and RNAs were purified and analyzed by denaturing PAGE and autoradiography. Positions of the injected RNAs as well as the intron lariat and spliced product of the ftz pre-mRNA are indicated.

The CRM1-NES-RanGTP complex is disassembled under the influence of RanBP1 and RanGAP. (A) Dissociation of a complex of CRM1, MVM NS2 NES, and RanGTP as a function of increasing concentrations of RanBP1 using the CRM1 GAP assay. The CRM1, MVM NS2 NES, RanGTP, and the RanGAP Rna1p were present at 1 μM, 4 μM, 100 nM, and 20 nM, respectively. (B) A trimeric complex that had been formed by binding of 0.5 μM CRM1 and 2 μM RanGTP to a GST-An3N column (equivalent to 1.5 μM) was incubated without (lane 1) or with (lanes 2 and 4) 0.05 μM RanBP1 and/or 0.25 μM Rna1p (lanes 3 and 4) for 5 min at room temperature, and bound (upper panel) and released (lower panel) CRM1 was analyzed by SDS-PAGE and silver staining.

Xenopus homologues of nucleoporins CAN and Nup88 specifically bind to CRM1 in a RanGTP-dependent, WGA-sensitive manner. (A) Proteins bound to a z-tagged CRM1 column from total Xenopus egg extract in the absence (lane 1) or presence (lanes 2 to 4) of 2 μM RanQ69LGTP and 10 μM MVM NS2 NES and in the presence or absence of 270 μg of WGA/ml and/or 250 mM N-acetylglucosamine (Glc-N-Ac) as indicated were analyzed by SDS–5 to 20% PAGE and silver staining. Positions of Xenopus CAN, Nup88, and RanQ69LGTP are indicated. (B) Proteins from Xenopus extract bound to a CRM1 column in the presence or absence of RanQ69LGTP and NES peptide (lanes 1 and 2) were compared with those bound to a z-tagged exportin t (XPO-t) column in the presence or absence of RanQ69LGTP (lanes 3 and 4). IgG-Sepharose (lane 5) served as an additional control. Analysis was as described for panel A, and positions of CAN, Nup88, RanQ69LGTP, and tRNA are indicated.

A model for directionally of CRM1-mediated nuclear export. In the nucleus, trimeric complex formation between CRM1, NES cargo, and RanGTP is promoted by high RanGTP concentration. This interaction can, however, be prevented by the cytotoxin leptomycin B (, ). The trimeric complex traverses the NPC, a process that is reversible under certain conditions (). In the cytoplasm RanBP1 or RanBP1-like domains in RanBP2-Nup358 destabilize the trimeric complex, a process that is made irreversible by GTP hydrolysis on Ran stimulated by RanGAP. Note that RanBP1 (or RanBP1-like domains) and RanGAP are present in the cytoplasm or on the cytoplasmic face of NPCs.